54 research outputs found
Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent
The soil actinomycete Kutzneria sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-N5-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD(P)H-dependent N-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes.National Center for Research Resources (U.S.) (P41RR012408)National Institute of General Medical Sciences (U.S.) (P41GM103473
Temperature and force dependence of nanoscale electron transport via the Cu protein Azurin
The mechanisms of solid-state electron transport (ETp) via a monolayer of
immobilized Azurin (Az) was examined by conducting probe atomic force
microscopy (CP-AFM), both as function of temperature (248 - 373K) and of
applied tip force (6-12 nN). By varying both temperature and force in CP-AFM,
we find that the ETp mechanism can alter with a change in the force applied via
the tip to the proteins. As the applied force increases, ETp via Az changes
from temperature-independent to thermally activated at high temperatures. This
is in contrast to the Cu-depleted form of Az (apo-Az), where increasing the
applied force causes only small quantitative effects, that fit with a decrease
in electrode spacing. At low force ETp via holo-Az is temperature-independent
and thermally activated via apo-Az. This observation agrees with
macroscopic-scale measurements, thus confirming that the difference in ETp
dependence on temperature between holo- and apo-Az is an inherent one that may
reflect a difference in rigidity between the two forms. An important
implication of these results, which depend on CP-AFM measurements over a
significant temperature range, is that for ETp measurements on floppy systems,
such as proteins, the stress applied to the sample should be kept constant or,
at least controlled during measurement.Comment: 24 pages, 6 figures, plus Supporting Information with 4 pages and 2
figure
Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase
Metals in Biotechnology: Cr‐Driven Stereoselective Reduction of Conjugated C=C Double Bonds
Substrate Binding and Reactivity Are Not Linked: Grafting a Proton-Transfer Network into a Class 1A Dihydroorotate Dehydrogenase
Mechanism of Dihydrouridine Synthase 2 from Yeast and the Importance of Modifications for Efficient tRNA Reduction*
Dihydrouridine synthases (DUSs) are flavin-dependent enzymes that catalyze
site-specific reduction of uracils in tRNAs. The mechanism of DUS 2 from
Saccharomyces cerevisiae was studied. Previously published turnover
rates for this DUS were very low. Our studies show that the catalytic cycle
consists of reductive and oxidative half-reactions. The enzyme is reduced by
NADPH rapidly but has a very slow oxidative half-reaction using in
vitro transcribed tRNA substrates. Using tRNALeu purified from
a DUS 2 knockout strain of yeast we obtained reaction rate enhancements of
600-fold over in vitro transcribed substrates, indicating that other
RNA modifications are required for rapid uracil reduction. This demonstrates a
previously unknown ordering of modifications and indicates that dihydrouridine
formation is a later step in tRNA maturation. We also show that an active site
cysteine is important for catalysis, likely in the protonation of uracil
during tRNA reduction. Dihydrouridine of modified tRNA from Escherichia
coli was also oxidized to uridine showing the reaction to be
reversible
Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis
Subunit activity and cooperativity of a homodimeric flavoenzyme, dihydroorotate dehydrogenase A (DHODA) from Lactococcus lactis, were characterized by employing single-molecule spectroscopy to follow the turnover kinetics of individual DHODA molecules, eliminating ensemble averaging. Because the enzyme-bound FMN is fluorescent in its oxidized state but not when reduced, a single DHODA molecule exhibits stepwise fluorescence changes during turnover, providing a signal to determine reaction kinetics and study cooperativity. Our results showed significant heterogeneity in the catalytic behaviors of individual dimer molecules, with only 40% interconverting between the three possible redox states: the fully fluorescent (both subunits oxidized), the half-fluorescent (one subunit oxidized and the other reduced), and the nonfluorescent (both subunits reduced). Forty percent of the single dimer traces showed turnovers between only the fully fluorescent and half-fluorescent states. The remaining 20% of the molecules interconverted only between the half-fluorescent state and the nonfluorescent state. Kinetic analysis revealed very similar reaction rates in both the reductive and oxidative half-reactions for different DHODA dimers. Our single-molecule data provide strong evidence for half-sites reactivity, in which only one subunit reacts at a time. The present study presents an effective way to explore the subunit catalytic activity and cooperativity of oligomeric enzymes by virtue of single-molecule fluorescence
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